How Lipid Membranes Affect Pore Forming Toxin Activity - Accounts of

Dec 7, 2015 - In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In g...
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How Lipid Membranes Affect Pore Forming Toxin Activity Nejc Rojko† and Gregor Anderluh*,†,‡ †

Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia



CONSPECTUS: Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure−function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein−lipid membrane interactions in greater detail.



Salmonella enterica strains is built with α-helices, thus belonging to α-PFTs (Figure 1C).3 Both, α-hemolysin and ClyA were the first representatives of their structural types with the solved structure of the pore. Transmembrane pore formation can, however, go beyond protein-lined channels and can also involve the participation of lipids, where the pore is built from the polypeptide chain and the lipid headgroups.4 There is considerable interest in understanding molecular mechanisms of PFTs, since they are important bacterial virulence factors and similar proteins have a prominent role in the vertebrate immune system by providing defense against pathogens and unwanted cells. PFTs are also a very good model for studying different aspects of transmembrane protein structure and function. Traditionally the research was focused on structure−function relationship, and we know much about the structural features of various PFT families, and in many cases molecular mechanisms responsible for specific toxin membrane binding or protomer−protomer interactions within

INTRODUCTION Plasma membrane defines cell boundaries and is involved in key biological processes. It is, therefore, on the front of pathogen attack and often serves as a primary target for secreted toxins. Toxins are designed to deliver toxic components across cellular membranes or directly damage lipid membrane and disrupt its integrity. A large number of socalled pore-forming toxins (PFTs) are able to achieve this by formation of transmembrane pores in lipid membranes.1 This enables the transport of components that is not regulated by cellular mechanisms and is detrimental for the cell. PFTs are in general synthesized as water-soluble monomeric species. After binding to the target membrane, they have to undergo a remarkable transformation from soluble form to membrane spanning structures that are tightly associated with the lipid bilayer (Figure 1A). Toxins that use β-barrels to form the pore wall are classified as β-PFTs; an archetypal example is Staphylococcus aureus α-hemolysin (Figure 1B), where seven α-hemolysin monomers contribute their β-hairpins to constitute the final transmembrane β-barrel.2 In contrast, the pore of cytolysin A (ClyA) produced by several Escherichia coli and © XXXX American Chemical Society

Received: September 3, 2015

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Figure 2. Lo domains and phase boundaries do not necessarily act as an additional concentration platform for promoting oligomerization. (A) Time sequence of fluorescently labeled EqtII binding to phase separated model membrane (droplet interface bilayer). Immediately after injection, EqtII preferentially binds the domain boundary as seen by the higher fluorescence intensity at the Lo/Ld border (white arrow) and is eventually associated with Ld domain (red asterisk). EqtII also remodels the domain shape and size (yellow asterisk denotes bulk Lo phase). Scale bar = 10 μm. (B) A series of images showing that EqtII forms pores in Ld phase of droplet interface bilayers, regardless of initial accumulation at phase boundaries. At the point of bilayer perforation, Ca2+ flux is visualized with Fluo-8H dye (green). Fluorescently labeled EqtII fluorescence is shown in red; dark area is Lo domain. Direction of events is denoted by the arrow; events from the first line are continued in the second line. Images were taken every 100 ms; scale bar = 5 μm. Adapted with permission from ref 9. Copyright 2014 Elsevier.

Figure 1. Despite considerable differences in structures, PFTs damage membranes by a similar multistep mechanism.1 (A) Toxin binds the target membrane (i), and monomers oligomerize (ii) and punch a hole through the membrane (iii) by conformational rearrangements of the polypeptide chain. Lipid membrane properties can affect this process at any step. (B) α-Hemolysin from Staphylococcus aureus is an archetypal representative of β-PFTs, where the transmembrane pore is a β-barrel. (C) ClyA from Escherichia coli forms a pore with a cluster of helices and thus belongs to α-PFTs. One protomer is highlighted in purple in each pore. The orange rectangle denotes the lipid membrane region, where the transmembrane part of the pore is placed.

the pore are well-defined.1−5 However, physicochemical properties of the lipid membrane can significantly impact the pore formation process. Membrane properties like separation of lipids in different domains, hydrophobic mismatch, and fluidity govern receptor availability, toxin oligomerization, and membrane penetration and can therefore affect also the properties of the final pores. In this Account, we focus on PFTs that interact more or less selectively with abundant membrane lipids such as phospholipids, including sphingomyelin (SM), and sterols and discuss the importance of the bulk membrane properties on the pore forming process.

phosphatidylcholine (PC) and SM, which both contain a phosphocholine headgroup, must occur deeper in the bilayer, it is possible that the tightly packed liquid ordered (Lo) lipid phase hampers lipid recognition. Numerous studies also show that addition of cholesterol (Chol) to SM containing membranes improves binding of actinoporins, even though Chol by itself is not recognized. This led to the suggestion that phase separation, promoted by Chol, exposes lipid molecules for easier binding due to packing defects at the phase boundary where EqtII was shown to accumulate.8−10 Another SM binding PFT, lysenin, is a defense-oriented β-PFT purified from the coelomic fluid of earthworms.11,12 While EqtII preferentially binds dispersed SM, lysenin exclusively binds clustered SM.7 It is therefore not surprising that lysenin and EqtII do not bind the same plasma membrane regions of cells7,13 (even though both are SM specific toxins). In this context, the term cluster of lipids describes small lateral aggregates composed of fewer than 10 lipid molecules.7 These nanoscale clusters (also called lipid rafts) are short-lived but can be stabilized to coalesce to larger lipid domains.14 Another β-PFT family, cholesterol dependent cytolysins (CDCs), uses Chol as a specific lipid receptor.15,16 The Chol− CDC interaction is enabled by amino acids located on one of the loops at the bottom of the C-terminal domain 4 (D4).17 Because Chol acts as a CDC receptor, it was generally believed that lipid rafts, where Chol is enriched, are the primary binding site. This was supported by the fact that a certain Chol threshold is needed for CDC high permeabilizing activity, that is, sigmoidal response on Chol concentration,18 and that CDCs colocalize with detergent-resistant membrane (DRM) fractions.19−21 Labeled D4 of perfringolysin (PFO) from Clostridium perf ringens was even suggested as a tool to detect cholesterol-rich rafts.22 However, extracting rafts by detergents



LIPID MEMBRANE AS A TARGET FOR PFTs BINDING PFTs evolved to target specific lipid membranes by binding to an exposed molecular components of the host cell surface, such as proteins, sugars, or lipids, and thus using them as specific receptors.5 Specific binding to exposed protein is usually straightforward, resembling binding by well-defined complementary surfaces, such as in antigen−antibody interactions. Protein−lipid interactions are more challenging to assess experimentally. This is nicely demonstrated with the example of PFTs that are able to specifically recognize sphingomyelin, which acts as a lipid receptor in the lipid membrane. Equinatoxin II (EqtII) belongs to the α-PFT actinoporin protein family present in sea anemone venom. Specific binding to SM was shown by several different in vitro assays, such as lipid dot blots, surface plasmon resonance, or ELISA.6,7 However, EqtII interaction with large unilamellar vesicles and lipid monolayers composed solely of SM, where lipids are tightly packed, is very poor.8 Accordingly, EqtII colocalizes with the liquid disordered (Ld) lipid phase as seen by fluorescent microscopy (Figure 2).7,9 Because differentiation between B

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oligomerization is the final step in actinoporin pore formation mechanism, EqtII is expected to oligomerize in Ld where pore formation was visualized.9 Similar to EqtII, GUVs imaging revealed native PFO also accumulated at the Lo/Ld domain border. On the other hand, nontransmembrane PFO, which is still allowed to oligomerize, did not show any domain colocalization preference, which puts membrane heterogeneity as promotor of oligomerization under the question.39 Altogether it appears that domains do not necessarily act as a concentration platform promoting PFTs oligomerization, even though the lipid receptor is enriched in particular lipid domain. One exception is lysenin, which oligomerizes in SM rich domains, because it recognizes only clustered SM, but how this affects oligomerization is not fully understood.40 Moreover, there is currently no data showing that domain boundaries promote PFTs oligomerization and accumulation at the phase boundary might be a nonspecific property of amphipathic proteins.41,42

at low temperature has met some serious criticism, giving different results depending on the concentration and type of detergent and duration and temperature of extraction, which even raised a question as to whether rafts are a real physiological phenomenon.23,24 In reality, Chol induces phase separation at a concentration below the threshold needed for high CDC activity, questioning the raft binding hypothesis.25 Chol in the membrane can be tightly associated with the lipid acyl chains or more exposed in the lipid/water interface. As the overall Chol concentration increases, this equilibrium can be shifted toward the exposed form, which is readily accessible for CDC binding, explaining the sigmoidal CDC response on Chol concentration.26 Chol availability for CDC binding is modulated also by the lipid headgroup dimensions together with acyl chain length and saturation.27 Accordingly, PFO does not bind SM/Chol enriched domains, where Chol is tightly associated with SM below the headgroup.25 This example demonstrates that Chol accessibility for CDC binding is governed by steric demands as a result of specific lipid interactions. Similarly, membrane organizational order affects the activity of another PFT, α-hemolysin, which binds and permeabilizes lipid vesicles containing lipids with the choline headgroup (PC, SM). Liposomes above phase transition temperature and liposomes with shorter and unsaturated lipids were more susceptible for permeabilization.28,29 It appears that lipids in the Ld phase are a more accessible binding partner also in the case of this archetypal β-PFT.30,31 All of these examples clearly demonstrate that lipid membrane receptor availability depends on the bilayer context and allowed us to draw a general conclusion regarding PFTs that bind specific lipid components. Headgroups of lipid molecules are the primary target of PFTs and are generally more exposed in more fluid bilayers. These domains therefore promote binding, even though some of the receptor lipids are more enriched in the rigid Lo phase. The situation is different in PFTs that evolved to recognize a precise lipid distribution pattern like SM clusters (lysenin) or SM/Chol complexes in uniform Lo phase, which are recognized by the aegerolysin protein family.32−34



MEMBRANE PENETRATION BY PFTs DEPENDS ON THE MEMBRANE PROPERTIES In most cases, the final step in pore formation is insertion into the membrane and transfer of α-helices or β-hairpins across the membrane bilayer. Here physical properties, such as membrane fluidity and hydrophobic mismatch, play an important role and can even affect the properties of final pores. For example, the binding of E. coli α-hemolysin to Lo phase does not automatically result in protein insertion and pore formation.43 In the case of actinoporins, it seems that Ld phase more readily accommodates N-terminal α-helix insertion.9,44 In agreement, lowering the order parameter of the membrane by incorporating different solvents decreases the energetic barrier for PFT insertion, including peptide melittin, EqtII, and α-hemolysin.45 Although lysenin generally binds SM in Lo phase,7 permeabilization of uniform ordered phase in GUVs is slower compared with that in phase separated membrane, even though binding and oligomerization are not hampered. This implies that the transition from the prepore to the transmembrane channel is enhanced in the presence of Ld phase.46 The importance of bilayer fluidity for the shape and properties of transmembrane pores was exemplified recently by studies of CDCs and the related MACPF domain protein perforin (PFN). CDCs and proteins containing MACPF domain are now classified in the same superfamily, collectively referred to as MACPF/CDCs superfamily of proteins.47,48 The common feature of both protein families is a domain with twisted β-sheet decorated on both parts with clusters of helices, which rearrange during the pore forming mechanism to contribute β-hairpins and build a large transmembrane βbarrel. Pores formed by MACPF/CDCs are the largest known and can range from approximately 15 nm in diameter for the case of PFN to more than 30 nm in case of CDCs. Oligomerization proceeds by the sequential addition of monomers to a growing arc-like intermediate until the full ring is formed.49 The pore-forming mechanism of MACPF/ CDCs involves a prepore intermediate, which presents an oligomerized ring assembly that sits atop the unperturbed membrane. It became apparent that the final step, the insertion of β-hairpins into the membrane, is governed by membrane fluidity. For example, in the case of PFN, more fluid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-diphytanoyl-sn-glycero-3-phosphocholine



LIPID MEMBRANES PROMOTE PFTs OLIGOMERIZATION At least in the case of β-PFTs, monomers first oligomerize and then transform from a membrane bound prepore state to a membrane inserted hydrophilic pore where assembly of protomers is a rate limiting process in pore formation.35,36 Fast membrane association and conformational change in contrast to slow oligomerization kinetics as shown by the stopped flow data led to a conclusion that oligomerization is a rate limiting step also in the case of α-PFT actinoporins.37 It was suggested that membrane microdomains play an important role as an additional concentrating platform, where toxin monomer collision rate is higher.38 Domain boundaries could also be involved in concentrating PFTs at the membrane plane. EqtII accumulated at the domain edges in phase-separated giant unilamellar vesicles (GUVs) and lipid monolayers as seen by fluorescence microscopy.8,10 Recently, microscopy of fluorescently labeled EqtII was combined with functional pore visualization in droplet interface bilayer. EqtII concentration at the phase boundaries was elevated, but surprisingly, functional channels were formed primarily in Ld phase where concentration is lower relative to the phase boundary, at least in the early stages of EqtII binding (Figure 2).9 Because C

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Figure 3. Insertion in the membrane and pore properties of MACPF/CDC proteins depends on the membrane lipid composition. (A) Different current traces from a planar lipid membrane experiment are presented, together with suggested shape of the pore. Pores are more noisy and smaller in more fluid lipid membranes, while nicely resolved pores with large conductance were visible in less fluid membranes. (B) Pore opening depends on the lipid membrane composition. In more fluid membranes pores were more often open gradually (unshaded regions), while in more ordered membranes more pores open in a discrete manner (shaded regions). Adapted with permission from ref 50. Copyright 2011 American Society for Biochemistry and Molecular Biology.

Figure 4. Partitioning of transmembrane proteins in different phases of model lipid membranes. (i, ii) Proteins with single transmembrane segment partition out of the Lo phase due to high energetic cost associated with penetrating highly ordered and thicker Lo phase. Proteins with a longer transmembrane segment are also driven out of the Lo phase, since they can easily tilt in the Ld phase thus avoiding a hydrophobic mismatch. (iii, iv) Partitioning of protein oligomers depends mostly on the hydrophobic mismatch. Tilting of large oligomers would expose hydrophobic segments and is thus prevented. Oligomers with longer transmembrane region partition into the thicker Lo phase, minimizing the hydrophobic mismatch and providing free energy for disrupting the Lo phase. Hydrophobic transmembrane segments are represented as cylinders; lipid molecules are depicted in orange and Chol in red.

observed perpendicular to the membrane plane, and negative curvature is present in the membrane plane around the pore. The presence of lipids that induce membrane curvature, therefore, promoted actinoporin55 and α-PFT colicin activity.56 Leakage from lipid vesicles in the case of melittin, a peptide forming toroidal pores, is also sensitive to lipid spontaneous curvature.57 Moreover, lipid molecules can directly influence electric conductive properties of the pore. Negatively charged lipids increase cation selectivity of EqtII pores, confirming lipid presence in the pore walls.58 Another important factor driving the distribution of large βbarrels in membranes is the hydrophobic mismatch between the barrel and the membrane lipid bilayer. Shorter barrels tend to insert into thinner membranes, including those with the Ld phase, and longer barrels tend to insert into thicker ones such as the Lo phase (Figure 4). In contrast, hydrophobic mismatch is believed not to be responsible for partitioning of proteins with a single transmembrane segment, because it can adapt to different membrane thickness simply by tilting, a principle applicable not only for PFTs.59,60 In this context, protein oligomerization, changing the effective lateral dimension of the pore, influences protein membrane distribution.60 Most likely,

(DPhPC) are easily penetrated by PFN; pore openings are observed even before PFN assembles a full ring prepore structure.50 This implies that pore walls are lined by lipids on the side where the ring is incomplete, thus forming a proteolipid (toroidal) pore.4 Because of the lipid presence, the pores are very noisy, as shown by measuring ionic currents in a planar lipid membranes setup (Figure 3). More ordered lipids like 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) do not support prompt penetration of incomplete PFN rings; therefore a stable pore opens in a clear discrete jump only after PFN assembles a full ring-shaped prepore.50 Complete PFN rings were also most frequent when Chol content was high, in agreement with its ordering effect on the lipid bilayer.51,52 A similar effect was observed for listeriolysin O (LLO), a CDC from the pathogenic bacterium Listeria monocytogenes. While LLO opens smaller and noisier pores in fluid membranes, such as DOPC/Chol mixtures,53 the pores are larger and less noisy in more ordered membranes, such as membranes composed of POPC/Chol in 1:1 ratio.54 Lipids can also affect other properties of pores when they form part of the pore wall, for example, in toroidal pore arrangement.4 In this pore, positive membrane curvature is D

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Most partitioning studies are performed in model membranes using phase-separated binary or ternary lipid mixtures and even lipids isolated from living cells. However, lateral organization in model membranes does not reproduce raft structure in cell membranes. Historically, DRM and Chol/SM depletion techniques were the basis for investigating raft affinity of lipids or proteins, but these techniques are invasive and may result in significant artifacts. Model membranes can serve as guidance in the understanding how proteins and lipids interact; however their behavior and fate in living cells must be investigated directly on the plasma membrane. Novel optical approaches capable of studying rafts beyond the diffraction limit and allowing imaging of single molecules and pores emerged in recent years.14,65,66 In the future, utilization of these techniques should help to reveal unprecedented details of PFT interactions with the membranes.

sorting between domains is driven by a combined effect of hydrophobic mismatch and bilayer fluidity. When protein or protein complex is too large to bury its hydrophobic segments in the Ld by tilting, the unfavorable hydrophobic mismatch could overcome the energetic barrier of complex inserting into Lo phase. Furthermore, if protein segments in complexes are not rigid, Lo domain could control the multiple conformation states to better fit the transmembrane segments.59



MEMBRANE ORGANIZATION REARRANGEMENTS AFTER PFT BINDING In many cases, PFTs do not just passively interact with the membrane but can influence its physical properties and organization. Actinoporins can increase the Lo size9 and coalesce lipid rafts in living cells, which could be an additional strategy of toxicity against the target cell.21,61 The key parameter determining the lateral membrane organization and domain sizes is the interfacial energy at the domain boundary, also known as the line tension.62 Based on the study of the proapoptotic pore forming protein Bax,63 a decreased line tension may be a general strategy of PFTs, because it stabilizes the open-pore state. The main factor causing the line tension is a hydrophobic mismatch between membrane phases.62 Strikingly, the actinoporins sticholysins reorganize the membrane and decrease a height mismatch between domains according to an atomic force microscopy study.64 LLO also induces lipid raft aggregation in the cell membrane. Because LLO binds Chol, which is enriched in lipid rafts, oligomerization may cross-link different nanoscale rafts resulting in their coalescence into visible domains.21 In contrast, lysenin neither binds the domain boundary nor decreases the line tension. Binding and oligomerization occurs in the SM rich Lo phase. Only after oligomers underwent prepore to pore conversion was the phase mixing to more uniform ordered phase observed, and this was suggested to be due to SM and Chol expulsion to the Ld phase.40



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The authors are grateful to Slovenian Research Agency for support of this research under Programme Grant “Molecular Interactions” P1-0391. Notes

The authors declare no competing financial interest. Biographies Nejc Rojko (b. 1985) holds a degree in biotechnology from Biotechnical Faculty, University of Ljubljana, where he also received his Ph.D. degree in 2013 studying biophysical and biochemical properties of pore forming proteins from sea anemones. At the moment, he is a postdoctoral researcher in the Laboratory for Molecular Biology and Nanobiotechnology at the National Institute of Chemistry in Ljubljana, Slovenia. His work involves studying the pore forming mechanism of new cnidarian pores and mechanisms involved in virus particle assembly.



CONCLUSIONS Membrane physicochemical properties have a large impact on assembly, shape, and properties of transmembrane pores formed by PFTs. Lipids are more available for binding in the Ld state, unless protein specifically recognizes organized features like lipids in clusters or a combination of different lipids. Phase boundaries might also be a preferential binding site due to better exposure of lipids, but it is not clear whether this facilitates binding, membrane insertion, or both. Lipid rafts were initially proposed to act as an oligomerization platform. Remarkably, even some proteins that bind components enriched in rafts (SM, Chol) show more biological activity in Ld phase, questioning the role of domains as the additional concentration platform. Moreover, the phase boundary, where some proteins accumulate, has not been directly shown to participate in toxin assembly. Membrane bulk properties have considerable impact on the ability of PFTs to insert and translocate transmembrane segments. This appears to be easier in more fluid membranes. When protein oligomerizes and forms a pore, hydrophobic mismatch might direct the complex either to Ld or to Lo, depending on the bilayer thickness and the length of the hydrophobic part of the protein. If the unfavorable hydrophobic mismatch between the complex and the Ld phase is too high, this provides free energy for insertion into the more rigid Lo phase.

Gregor Anderluh (b. 1969) is head of the Laboratory for Molecular Biology and Nanobiotechnology at the National Institute of Chemistry, Slovenia, and professor of Biochemistry at the University of Ljubljana, Slovenia. He received a B.S. degree in biology in 1994 and Ph.D. in biology in 1998, both from the University of Ljubljana. He did his postdoctoral studies at the University of Newcastle upon Tyne, United Kingdom. He is also head of the Infrastructural Centre for Molecular Interaction Analysis at the University of Ljubljana, where they study molecular interactions and are developing novel approaches for studying protein binding to membranes. He and his co-workers are studying protein−membrane interactions and how cellular membranes are damaged by proteins in bacterial pathogenesis and the immune system.



ACKNOWLEDGMENTS We thank to Prof. Peter Maček for critical reading of the manuscript.



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